The present invention relates to a method of diagnosing, classifying and grading of tumor growths and to determine whether the use of chimeric polioviruses is a proper course for the treatment of the tumors. More particularly, the method is directed to the use of antibodies to a poliovirus receptor (PVR), CD155, to detect the presence of CD155 on tumor cells in various organs, such as: breast, colon, bronchial passage, epithelial lining of the gastrointestinal, upper respiratory and genito-urinary tracts, liver, prostate and the brain.
It has been found by the inventors that malignant tumors can be successfully treated with chimeric polioviruses wherein the internal ribosomal entry site (IRES) is replaced with the IRES of another picornavirus. This finding is the subject of U.S. patent application Ser. No. 09/129,686, incorporated herein by reference.
Poliovirus is a small iscosahedral RNA virus belonging to the picornavirus family. It is best known as the etiologic agent of poliomyelitis, the visible clinical sign of which is flaccid paralysis. The virus when ingested, infects and replicates in the gut, leading to viremia. In a small portion of infected individuals, the virus invades the central nervous system through the circulation. It is the lytic replication in motor neurons of the brain stem and spinal cord that causes destruction of these cells and the characteristic flaccid paralysis of poliomyelitis. Bodian D., Science (1955)12:105-108. It is known that poliovirus invades only a limited number of specific cells in the body, an unknown population of cells lining the gastrointestinal tract and spinal cord anterior horn as well as medullary motor neurons. It is the unusual restricted cell tropism of poliovirus that leads to unique pathognomonic features, characterized by motor neuron loss in the spinal cord and the medulla, giving rise to the hallmark clinical sign of poliomyelitis, flaccid paralysis, Bodian, D., supra. The restricted tropism of poliovirus is poorly understood. In addition to the restricted cell and tissue tropism, poliovirus only infects primates and primate cell cultures. Other mammalian species remain unaffected. Ren, R. et al., Cell (1990) 63:353-362.
The isolation of poliovirus in 1908 led to intensive research efforts to understand the mechanisms of infection. The earlier work required the use of monkeys and chimpanzees as animal models. Such animals with longer life cycles are very costly and difficult to use in research. The discovery of the human poliovirus receptor (PVR), the cellular docking molecule for poliovirus, led to the development of a transgenic mouse expressing the human poliovirus receptor as a new animal model for poliomyelitis. Mice transgenic for PVR, when infected with poliovirus, develop a neurological syndrome histopathologically and clinically identical to primate poliomyelitis. Koike et al., Proc. Natl. Acad. Sci. U.S.A. (1991) 88:951-955. The observation of poliomyelitis in polio-infected transgenic animals suggests that CD155 alone is responsible for directing poliovirus towards spinal cord anterior horn motor neurons mediating their infection and subsequent lytic destruction. Gromeier et al., Proc. Natl. Acad. Sci. U.S.A. (1996) 93:2370-2375. The poliovirus receptor, previously referred to as PVR, has now been classified as CD155. Mendelsohn C. et al., Cell (1989) 56:855-865 and U.S. Pat. Nos. 5,631,407 and 5,753,521.
Up to the present, CD155 has not been associated with malignant tumors. Polypeptide CD155 is a cell-surface protein belonging to the immunoglobulin superfamily. Its gene is the founding member of a new family of primate and rodent genes that encode polypeptides with a common structural arrangement of three extracellular (V-C2-C2) domains. CD155 is expressed in four isoforms: hPVRxcex1 and hPVRxcex4 are membrane-bound variants that differ only in the sequence of the cell-internal C-terminal domain, while hPVRxcex2 and hPVRxcex3 are secreted isoforms lacking the transmembrane domain. Koike et al., EMBO J. (1990) 9: 3217-3229; Wimmer et al., In: Cellular Receptors for Animal Viruses (1993) Cold Spring Laboratory Press: Plainview. N.Y. hPVRxcex1 and hPVRxcex4 are type Ia single-pass transmembrane glycoproteins with apparent Mr of  greater than 80 kDa, whereas the core polypeptides are 42.5 and 40 kDa, respectively. Binding of poliovirus occurs at the V-domain of the polypeptide. Bernhardt et al., Virology (1994) 203: 344-356.
First indications for an association between CD155 and cancer are based on assays making use of polioviruses to infect and destroy tumor cells derived from human malignancies. In co-pending U.S. application Ser. No. 09/129,686, the effectiveness of recombinant chimeric polioviruses for the treatment of various forms of cancer was desribed. Human tumor cells obtained from explant surgical material were susceptible to poliovirus whereas their non-malignant progenitors are known to resist poliovirus infection. It was suspected that the acquisition of susceptibility to poliovirus is based on the overexpression of the cellular receptor for poliovirus on cells upon malignant transformation. A precedent for this assumption is provided by a homolog of CD155 in rodents, the murine Tage4 molecule. Tage4 was isolated from rodent colon- and mammary carcinomas, where it occurs in abundance. Chadeneau et al., Int. J. Cancer (1994) 68:817-821. In contrast, Tage4 could hardly be detected in normal rodent colon- or mammary duct epithelium. Chadeneau et al., supra.
The link of CD155 with cancer is further evident from studies of the expression of CD155 during embryonic development. Like many of its fellow members of the immunoglobulin superfamily, CD155 appears to be expressed during embryonic development. Frequently, immunoglobulin superfamily molecules that are expressed in a developmental manner have been associated with malignancy. It has been determined that CD155 may be expressed in a group of highly specialized structures within the embryonic central nervous system, e.g., floor plate, notochord, and optic nerve. These studies not only provided evidence for the distribution of CD155 in the developing nervous system but also pointed towards a physiological function of CD155 that may encompass a role during morphogenesis of the central nervous system. Molecules related to CD155 in structure or sequence are known to be expressed with an overlapping distribution during central nervous system ontogeny. Walsh et al., Annul Rev. Cell Dev. Biol. (1997) 13:425-456; Colamarino et al., Annul Rev. Neurosci. (1995) 18:497-529.
Molecules of the immunoglobulin superfamily, for example Tage4 (see above), are increasingly recognized for their association with cancer. Sasaki et al., Cancer (1998) 82:1921-1931; Gingras et al., J. Neuroimmunol. (1995) 57: 143-153; Figarella-Branger et al., Cancer Res. (1990) 50:6364-6370. It is believed that molecules belonging to the immunoglobulin superfamily with a function involving the mediation of cell adhesion and axonal guidance are frequently overexpressed in malignant tumors. Their physiological functions may be of relevance for the biology of tumors. Aberrant properties of tumor cells, for example invasiveness, migration and metastatic spread may correlate to the expression of immunoglobulin superfamily molecules with a function in cell adhesion. Izumoto et al., Cancer Res. (1996) 56:1440-1444. Corroborating the findings with other cell adhesion molecules of the immunoglobulin superfamily with a role in embryonic development of the CNS we provided extensive evidence that CD155, indeed, is abundant in a variety of human malignancies (see below).
Antibodies to CD155
Monoclonal antibodies which recognize CD155 have been developed. In particular, a murine monoclonal antibody was developed by immunizing mice with plasma membrane preparations of HeLa cells. The monoclonal antibody D171 was selected for its ability to protect HeLa cells against the cytopathic effect of poliovirus. It was found that the antibody or its Fab fragments bound to cell lines of human or primate origin and such binding can be blocked after pre-incubation with poliovirus. Nobis, P. et al. J. Gen. Virol., (1985) 66:2563-2569. HeLa cell membrane preparations were used in order to raise monoclonal antibodies against the receptor for poliovirus because they were the most commonly available cell line to propagate poliovirus in tissue culture. HeLa cells are derived from a human cervix carcinoma. Carcinoma cell lines that had been adapted to tissue culture through many hundred passages are commonly used to propagate poliovirus. The ability of these cell lines to efficiently replicate poliovirus implies that they must express CD155. However, the relation of CD155 with cancer was not obvious. Carcinoma cell lines (such as HeLa cells) routinely used for tissue culture purposes may have little in common with the tumor cells from which they were originally derived. An association of CD155 with human malignancies was first recognized by the inventors. This association only became evident after direct immunological probing for CD155 in human surgical tumor explants. The approach employed assured that expression levels of CD155 measured actually correspond to those in tumors found in cancer patients, rather than in tissue culture cell lines established decades ago that have been passaged innumerable times, e.g., HeLa cells.
Recombinant Polioviruses
Chimeric polioviruses carrying heterologus IRES elements, which have lost their inherent neuropathogenic potential have been described. Gromeier, M. et al., supra. Mice transgenic for PVR, when infected with poliovirus, develop a neurological syndrome histopathologically and clinically identical to primate poliomyelitis. Koike et al., Proc. Natl. Acad. Sci. U.S.A. (1991) 88:951-955. The observation of poliomyelitis in polio-infected transgenic animals suggests that CD55 alone is responsible for directing poliovirus towards spinal cord anterior horn motor neurons mediating their infection and subsequent lytic destruction. Gromeier et al., Proc. Natl. Acad. Sci. U.S.A. (1996) 93:2370-2375. Gromeier et al., J. Virol. (1999) 73:5056-5060 incorporated herein by reference. It was found that the substitution of the cognate IRES of poliovirus with its counterpart from Human Rhinovirus type 2 (HRV2) eliminated the ability of the resulting chimera, PV1 (RIPO) to grow within cells of neuronal derivation. The non-pathogenic phenotype of PV1 (RIPO) and PV1 (RIPOS) was documented in mice transgenic for the human poliovirus receptor, CD155 tg mice as well as primates. See Gromeier et al., supra. Despite its inability to replicate efficiently within normal cells of neuronal origin, it is now shown that PV1 (RIPO) retained wild-type growth characteristics with an ability to lyse tumor cells in a panel of rapidly dividing malignant cell types originating from human malignancies. The types of malignancies which responded to treatment by the chimeric polioviruses included: malignant gliomas, mammary carcinoma, colorectal carcinoma, hepatocellular carcinoma, bronchial carcinoma and epidermoid carcinoma. It is suspected that the malignant cells carry the CD155 receptor to permit the entry of the recombinant chimeric polioviruses to enter and cause cytolysis of the tumor cells.
Methods of Detecting Cancer-associated Proteins
The diagnosis, classification, and characterization of tumors according to factors expressed within the tumor cell cytoplasm or on the cell surface is well established in medical oncology. Frequently, ectopic or excessive expression of molecules not ordinarily found in normal cells occurs upon malignant transformation of cells of certain organs. This principle can be used for the diagnosis, detection, differential classification, grading and staging of tumors as well as a rationale for therapeutic approaches. Detection of cell adhesion molecules belonging to the immunoglobulin superfamily has been developed as a diagnostic and classifying marker, Sasaki et al., supra. Several commonly employed assays based on immunogenic detection of specific antigens present in human tissues have been used for this purpose. For example, immunohistochemistry is being used routinely in clinical diagnostic laboratories for the classification of tumors. For this purpose, surgically excised tumor samples were embedded rapidly in a freezing compound comprising water soluble glycol and resins matrix for cryostat sectioning (Tissue Tek OCT(trademark)). Frozen sections of 5 xcexcm thickness were made, mounted on silane coated glass slides and air dried at room temperature. The sections were fixed in acetone. The sections were treated with antibody to the specific CAM, washed and incubated with secondary anti-species antibody conjugated with horseradish peroxidase. Sections were washed thoroughly and finally incubated in a solution containing 0.6% hydrogen peroxide and the substrate diaminobenzidine tetrahydrochloride. The average staining intensity was evaluated and a comparison between the tumor sections and normal tissue sections was made. Gingras, M. -C. et al. J. Neuroimmunol., (1995) 57:143-153; Tsuzuki, T. et al. J. Clin. Pathol., (1998) 51:13-17. Western blot analysis has also been employed to study the presence of neural CAM in astrocytomas. Tissue in Tris-HCl buffer pH 7.4 was homogenized and centrifuged. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The blots were incubated overnight with anti-NCAM antibody ERIC-1 and visualized by an indirect secondary antibody method as described above. Blots were treated with an anti-species antibody recognizing ERIC-1 that was conjugated to horseradish peroxidase. In a staining reaction with a peroxidase substrate bands specific for NCAM could be identified. Sasaki, H. et al. Cancer, (1998) 82:1921-1931; Izumoto, S. et al., Cancer Res., (1996) 56:1440-1444.
It is an objective of the present invention to develop a method to detect the presence of CD155 in human cancers.
It is another objective of the present invention to be able to use the results obtained to determine whether the use of chimeric oncolytic polioviruses would be effective for treatment of malignancy if found.
A further objective of the present invention is to develop analytical methods employing antibodies to CD155 to detect the presence of CD155 to determine the degree of malignancy and to determine whether the use of chimeric poliovirus was appropriate for the treatment of the tumor.
1. Bodian D., Science (1955)12:105-108.
2. Ren, R. et al., Cell (1990) 63:353-362.
3. Koike et al., Proc. Natl. Acad. Sci. U.S.A. (1991) 88:951-955.
4. Gromeier et al., Proc. Natl. Acad. Sci. U.S.A. (1996) 93:2370-2375.
5. Mendelsohn C. et al., Proc. Natl. Acad. Sci. USA (1986) 83:7845-7849.
6. Koike et al., EMBO J. (1990) 9: 3217-3229.
7. Wimmer et al., In: Cellular Receptors for Animal Viruses (1993) Cold Spring Laboratory Press: Plainview. N.Y.
8. U.S. Pat. No. 5,631,407.
9. U.S. Pat. No. 5,753,521.
10. Bernhardt et al., Virology (1994) 203: 344-356.
11. Chadeneau et al., Int. J. Cancer (1994) 68:817-821.
12. Walsh et al., Annul Rev. Cell Dev. Biol. (1997) 13:425-456.
13. Colamarino et al., Annul Rev. Neurosci. (1995) 18:497-529.
14. Nobis, P. et al. J. Gen. Virol., (1985) 66:2563-2569.
15. Gromeier, M. et al., J. Virol., (1999) 73:5056-60
16. Gingras, M. -C. et al. J. Neuroimmunol., (1995) 57:143-153.
17. Tsuzuki, T. et al. J. Clin. Pathol., (1998) 51:13-17.
18. Sasaki, H. et al. Cancer, (1998) 82:1921-1931.
19. Figarella-Branger et al., Cancer Res. (1990) 50:6364-6370.
20. Izumoto, S. et al., Cancer Res., (1996) 56:1440-1444.
According to the present invention a method of determining the malignancy of tumor tissue and determining whether the use of recombinant chimeric poliovirus for treatment of the malignant tumor is appropriate has been developed. The method comprises the steps:
a) treating surgical explant tumor tissue to prepare a thin section of the tumor tissue for mounting the section on a pre-coated microscopic slide;
b) fixing and blocking the section on the slide;
c) reacting the section with an anti-CD155 monoclonal antibody to bind CD155;
d) reacting a secondary antibody conjugated to a chromophore with the bound anti-CD155 to form a complex;
e) developing a color by reacting the complex with a chromogenic substrate for the chromophore.
The tumor tissue may be prepared by either shock freezing or embedding in paraffin. Thin cryosections or paraffin-embedded sections of the tumor tissue are prepared and mounted on silane-coated microscope slides. The sections are fixed on the slides with fixing agents, such as: paraformaldehyde in phosphate buffered saline (PBS), glutaraldehyde in PBS, and acetone. The fixed tumor tissue sample is incubated from about 2 to 10 min. For detection of antibody reactivity the substrates for the chromophore may include chromogenic, fluorescent or chemiluminescent agents. Chromogenic substrates include agents such as 5-bromo-4-chloro-3-indolyl-phosphate 4 toluidine/nitro blue tetrazolium chloride for alkaline phosphatase and 3-amino-9-ethyl-carbazole for horseradish peroxidase; fluorescent substrates like 2-hydroxy-3-naphthoic acid-2xe2x80x2-phenylanilide phosphate/4-chloro-2-methylbenzene-diazonium hemizinc chloride for alkaline phosphatase; chemiluminescent substrates like disodium 4-chloro-3-(methoxyspiro {1,2-dioxetane-3,2xe2x80x2-(5xe2x80x2-chloro) tricyclo [3.3.1.13,7] decan}-4-yl)phenyl phosphate for alkaline phosphate and luminol/4-iodophenol for horseradish peroxidase.
Alternatively the tumor tissue samples may be homogenized in a suitable solubilization buffer. After centrifuging and filtering to remove debris, the protein concentration of the supernatant fluid is determined and adjusted to a standard selected for all tumor samples. Then the supernatant fluid is boiled for 5 min. in a buffer, such as Laemmli buffer, and subjected to sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). The separated proteins are Western blotted, and treated with a monoclonal anti-CD155 antibody and secondary antibody conjugated to a chromophore as described above selected to visualize the protein bands specific for CD155.
The proteins in the supernatant liquid may also be immuno-precipitated, mixed with sepharose A, equilibrated in PBS, and then mixed with ascites fluid containing a monoclonal antibody to CD155 and incubated over night. The immunoprecipitate is then boiled in Laemmli buffer and the remaining steps of the procedure as described above are followed.
The above procedures may be also modified as an immunodot assay. The tumor samples are determined at the same time as a sample that is not tumorous. By comparison, the overexpression of CD155 can be determined. According to the present invention a method for determining the expression levels of the immunoglobulin superfamily molecule CD155 has been developed. Determination of the expression levels of CD155 can be used to classify, characterize and stage malignant tumors and may provide a rationale for the use of attenuated oncolytic poliovirus as therapy.